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Measurement of Protein-Protein Interactions through Microscale Thermophoresis (MST)

Authors: Magnez Romain, Bryan Thiroux, Morgane Tardy, Bruno Quesnel and Xavier Thuru, date: 04/05/2020, view: 62, Q&A: 0

[Abstract] The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations resulted in controversy regarding the values of the dissociation constant (K_d) (Freeman et al., 2000). MST is a powerful new method for the ...

Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation

Authors: Claudia Cattoglio, Iryna Pustova, Xavier Darzacq, Robert Tjian and Anders S. Hansen, date: 02/20/2020, view: 652, Q&A: 0

[Abstract] Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins ...

Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1

Authors: Wai Wa Ray Chan, Dik Long Dennis Chau, Wen Li and Kwok-Fai Lau, date: 12/05/2019, view: 604, Q&A: 0

[Abstract] Intramolecular interaction is a common mechanism that regulates protein activities. Conventionally, such interactions are investigated by classical in vitro biochemical assays. Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation ...

Detection of in vivo Protein Interactions in All Bacterial Compartments by Förster Resonance Energy Transfer with the Superfolder mTurquoise2 ox-mNeongreen FRET Pair

Authors: Nils Y. Meiresonne, Elisa Consoli, Laureen M.Y. Mertens and Tanneke den Blaauwen, date: 12/05/2019, view: 786, Q&A: 0

[Abstract] This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in all compartments of Escherichia coli by Förster Resonance Energy Transfer (FRET) using the Superfolder mTurquoise2 ox-mNeonGreen FRET pair (sfTq2^ox-mNG). This FRET pair has more than twice the detection range for FRET ...

Measuring Small-molecule Inhibition of Protein Interactions in Live Cells Using FLIM-FRET

Authors: James M. Pemberton, Qian Liu and David W. Andrews, date: 10/20/2019, view: 2172, Q&A: 0

[Abstract] This protocol was designed to quantitatively measure small-molecule displacement of proteins in live mammalian cells using fluorescence lifetime imaging microscopy–Förster resonance energy transfer (FLIM-FRET). Tumour cell survival is often dependent on anti-apoptotic proteins, which bind to and inhibit pro-apoptotic proteins, thus preventing ...

An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein

Authors: Yili Zhu, Weimin Tan and Wei-Lih Lee, date: 12/05/2018, view: 2440, Q&A: 0

[Abstract] In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by ...

FRET-based Stoichiometry Measurements of Protein Complexes in vitro

Authors: Francesca Mattiroli, Yajie Gu and Karolin Luger, date: 02/05/2018, view: 4375, Q&A: 0

[Abstract] For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro. This assay, also known as Job plot, is set up as a continuous variation of the ...

Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer

Authors: Nils Y. Meiresonne, Svetlana Alexeeva, René van der Ploeg and Tanneke den Blaauwen, date: 01/20/2018, view: 5397, Q&A: 0

[Abstract] This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Förster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent ...

Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay

Authors: Ana Lechuga, Mónica Berjón-Otero, Margarita Salas and Modesto Redrejo-Rodríguez, date: 01/05/2018, view: 5138, Q&A: 0

[Abstract] This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules ...

Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions

Authors: Yohei Katoh, Kentaro Nakamura and Kazuhisa Nakayama, date: 01/05/2018, view: 6196, Q&A: 0

[Abstract] The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can ...

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